The filoviruses, Ebola and Marburg viruses (EBOV and MARV), are emerging, highly virulent negative-strand RNA viruses classified as NIAID category A priority pathogens. Currently, no approved anti-filovirus therapeutics are available, in part due to the paucity of detailed knowledge of viral components, including knowledge of traditional targets such as the viral polymerase. Among the proteins encoded by EBOV, VP35 is of particular interest for antiviral development because it is well conserved among filoviruses, and it makes a critical contribution to pathogenesis through its role in viral RNA replication and in host immune suppression. The goal of this collaborative project is to develop lead compounds that act as pan-filoviral inhibitors of replication by targeting conserved, functionally critical regions of VP35. The approach builds upon the work of the Basler and Amarsinghe laboratories which has defined structural requirements for VP35 interferonantagonist and polymerase co-factor functions. Using this available data, the research team has used in silco approaches to identify small molecules that bind VP35 in close proximity to residues and structural features essential for both of these critical VP35 functions. These molecules bind VP35 as predicted and show inhibitory activity toward the viral polymerase complex in cell based assays. Initial SAR studies with available analogs have outlined structural requirements for binding. Based on solved structures of VP35-inhibitor cocrystals, the investigators have identified key features that determine inhibitor binding to VP35. Using the framework provided by this strong preliminary data we will establish a medicinal chemistry program involving synthetic chemistry (Dr. Ready), structural biology (Dr. Amarsinghe) and virology (Dr. Basler). The assembled collaborative research team will optimize and validate initial lead molecules into highly potent replication inhibitors with antiviral efficacy against filoviruses in cell culture and mice. To achieve these goals, the investigators will first synthesize optimized compounds with enhanced stability and efficacy. These efforts will be guided by biochemical and structural studies to evaluate VP35&#150;inhibitor interactions. These studies will facilitate the chemistry efforts of the first aim by characterizing VP35 IID-inhibitor interactions to assess affinity, specificity and structural complimentarity toward IFN inhibitory domains (IIDs) from different filoviruses. Those inhibitors exhibiting enhanced activity in vitro will be functionally assessed and validated in cell-based assays of VP35 function and anti-filoviral assays. Ultimately, optimized inhibitors exhibiting significant antiviral activity in cell culture will be assessed for antiviral activity in mice. To identify lead compounds with the greatest potential for drug development, preclinical toxicology, ADME and pharmacokinetic studies will be performed. At the completion of these studies, it is expected that highly potent, drug-like lead compounds will be available for further development.